P. pastoris pastoris promoters, and the use thereof to direct expression of proteins in yeast, preferably using a haploid mating strategy

ABSTRACT

Novel promoters which are derived from  P. pastoris pastoris  which are inducible or repressible under specific growth conditions are provided. These promoters are useful for regulating the expression of a desired structural gene, e.g., a mammalian polypeptide. Particularly preferred is the use of these novel promoters to regulate gene expression in polyploidal yeast such as diploidal  P. pastoris  produced by mating or spheroplast fusion.

This application is a Divisional of U.S. Ser. No. 13/542,851, filed Jul.6, 2012, which is a Divisional of U.S. Ser. No. 11/870,180, filed Oct.10, 2007, which claims priority to U.S. Provisional Application No.60/850,258, filed Oct. 10, 2006. The disclosure of each of theafore-mentioned provisional and non-provisional application includingall the sequence information is incorporated by reference in itsentirety herein.

BACKGROUND OF THE INVENTION Sequence Disclosure

This application includes as part of its disclosure a biologicalsequence listing text file named “43257o1404.txt” which was created onJul. 2, 2014 and has a size of 15,026 bytes, which is herebyincorporated by reference in its entirety.

Recombinant protein production is an essential activity for highthroughput screening, functional validation, structural biology, andproduction of pharmaceutical polypeptides. Escherichia coli is a widelyused organism for the expression of heterologous proteins because iteasily grows to a high cell density on inexpensive substrates, and haswell-established genetic techniques and expression vectors. However,this is not always sufficient for the efficient production of activebiomolecules. In order to be biologically active, polypeptide chainshave to fold into the correct native three-dimensional structure,including the appropriate formation of disulfide bonds, and may furtherrequire correct association of multiple chains.

Although the active state of the protein may be thermodynamicallyfavored, the time-scale for folding can vary from milliseconds to days.Kinetic barriers are introduced, for example, by the need for alignmentof subunits and sub-domains. And particularly with eukaryotic proteins,covalent reactions must take place for the correctly folded protein toform. The latter types of reaction include disulfide bond formation,cis/trans isomerization of the polypeptide chain around proline peptidebonds, preprotein processing and the ligation of prosthetic groups.These kinetic limitations can result in the accumulation of partiallyfolded intermediates that contain exposed hydrophobic ‘sticky’ surfacesthat promote self-association and formation of aggregates.

Recombinant synthesis of such complex proteins has had to rely on highereukaryotic tissue culture-based systems for biologically activematerial. However, mammalian tissue culture based production systems aresignificantly more expensive and complicated than microbial fermentationmethods. In addition, there continues to be questions regardingtherapeutic products produced using materials derived from animalby-products.

As a eukaryote, P. pastoris pastoris has many of the advantages ofhigher eukaryotic expression systems such as protein processing, proteinfolding, and posttranslational modification, while being as easy tomanipulate as E. coli or Saccharomyces cerevisiae. It is faster, easier,and less expensive to use than other eukaryotic expression systems suchas baculovirus or mammalian tissue culture, and generally gives higherexpression levels. As a yeast, it shares the advantages of molecular andgenetic manipulations with Saccharomyces. These features make P.pastoris very useful as a protein expression system.

Many of the techniques developed for Saccharomyces may be applied to P.pastoris. These include transformation by complementation; genedisruption and gene replacement. In addition, the genetic nomenclatureused for Saccharomyces has been applied to P. pastoris. There is alsocross-complementation between gene products in both Saccharomyces and P.pastoris. Several wild-type genes from Saccharomyces complementcomparable mutant genes in P. pastoris.

Heterologous expression in P. pastoris pastoris can be eitherintracellular or secreted. Secretion requires the presence of a signalsequence on the expressed protein to target it to the secretory pathway.While several different secretion signal sequences have been usedsuccessfully, including the native secretion signal present on someheterologous proteins, success has been variable. A potential advantageto secretion of heterologous proteins is that P. pastoris pastorissecretes very low levels of native proteins. That, combined with thevery low amount of protein in the minimal P. pastoris growth medium,means that the secreted heterologous protein comprises the vast majorityof the total protein in the medium and simple removal of the yeast cellsserves as the first step in purification of the protein.

Many species of yeast, including P. pastoris, are mating competent. Thisenables two distinct haploid strains to mate naturally and generate adiploid species possessing two complete sets of chromosomal copies.

Although P. pastoris has been used successfully for the production ofvarious heterologous proteins, e.g., hepatitis B surface antigen (Cregget aL (1987) Bio/Technology 5:479), lysozyme and invertase (Digan et al.(1988) Dev. Indust. Micro. 29:59; Tschopp et al. (1987) Bio/Technology5:1305), endeavors to produce other heterologous gene products in P.pastoris, especially by secretion, have given mixed results. At thepresent level of understanding of the P. pastoris expression system, itis unpredictable whether a given gene can be expressed to an appreciablelevel in this yeast or whether P. pastoris will tolerate the presence ofthe recombinant gene product in its cells. Further, it is especiallydifficult to foresee if a particular protein will be secreted by P.pastoris, and if it is, at what efficiency.

Various promoters have been derived from P. pastoris pastoris and usedto regulate the expression of homologous and heterologous proteins inyeast. These promoters include in particular the alcohol oxidasepromoters from the AOX1, AOX2 and mutant forms thereof as well as apromoter derived from the formaldehyde dehydrogenase gene FLD1. However,novel P. pastoris promoters especially P. pastoris promoters that areinducible or repressible under specific conditions and/or which providefor high expression yields in different yeast species are still needed.

The present invention satisfies this need and provides novel induciblepromoters which are derived from P. pastoris pastoris and methods of usethereof to regulate the expression of structural genes operably linkedthereto. In a preferred embodiment these promoters are used in thesubject Assignee's proprietary improved methods and expression vectorsthat provide for the secretion of heterologous proteins, especiallyheteromultimers, from mating competent yeast, desirably polyploid yeastand most preferably diploid P. pastoris strains.

SUMMARY OF INVENTION

The invention provides novel inducible promoters derived from P.pastoris pastoris. In particular the invention provides the nucleic acidsequences for the ADH1, ENO1 and GUT1 genes of P. pastoris pastoris.

Also, the invention provides DNA constructs wherein these novel P.pastoris pastoris promoters or mutant, hybrid or chimeric promotersderived therefrom are operably linked to one or more structural genes,preferably structural genes encoding a multichain mammalian protein suchas an immunoglobulin.

Also, the invention provides expression vectors, including autonomouslyreplicating plasmids and vectors that integrate into a yeast'schromosomal DNA randomly or site specifically containing at least onenovel yeast promoter according to the invention.

Further the invention provides transformed yeast, preferably transformedpolyploidal strains containing said expression vectors.

Still further the invention provides methods of expressing proteinsunder specific regulated or inducible conditions by expressing a geneencoding said protein under the regulatory control of a novel promoteraccording to the invention under conditions that favor expression.

In addition, methods are provided for the synthesis and secretion ofrecombinant hetero-multimeric proteins in mating competent yeast whereinthese hetero-multimeric proteins are expressed under the regulatorycontrol of a novel P. pastoris pastoris promoter according to theinvention. Hetero-multimeric proteins of special interest will compriseat least two non-identical polypeptide chains, e.g. antibody heavy andlight chains, MHC alpha and beta chains; and the like. An expressionvector is provided for each non-identical polypeptide chain whereineither or both contain a P. pastoris promoter according to theinvention.

Each expression vector is transformed into a haploid yeast cell. In someembodiments of the invention, the haploid yeast cell is geneticallymarked, where the haploid yeast cell is one of a complementary pair. Afirst expression vector is transformed into one haploid cell and asecond expression vector is transformed into a second haploid cell.Where the haploid cells are to be mated this will be through directgenetic fusion, or a similar event is induced with spheroplast fusion.

The expression levels of the non-identical polypeptides in the haploidcells may be individually calibrated, and adjusted through appropriateselection, vector copy number, promoter strength and/or induction andthe like. In one embodiment of the invention, the promoter in eachexpression vector is different. In another embodiment of the invention,the same promoter is provided for each. The novel promoters providedherein are inducible, i.e., they are transcriptionally active or “on”only under specific carbon source conditions.

In this preferred embodiment, the transformed haploid cells, eachindividually synthesizing a non-identical polypeptide, are identifiedand then genetically crossed or fused. The resulting diploid strains areutilized to produce and secrete fully assembled and biologicallyfunctional hetero-multimeric protein. The diploid methodology allowsoptimized subunit pairing to enhance full-length product generation andsecretion. However, the subject novel promoters may alternatively beused in conventional (non-polyploidal) yeast expression methods as well.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. Generation of assembled full length recombinant antibody.Immunoblot detection methodology was used to characterize the parentalhaploid P. pastoris strains, each producing a subunit of the antibodyand target diploid strain producing both subunits that form the fullyassembled antibody. The yeast strains shown in FIG. 1A show a staticculture of each of the representative strains, where the top portion isthe distinct haploids strains containing Heavy (H) and Light (L) chainsubunits respectively; the bottom the mated stable diploid producingboth subunits. FIG. 1B shows selective detection of the H chain, whichis found only in the parental H chain haploid, and mated diploidcontaining both H and L. FIG. 1C shows general detection of H and Lchains, which establishes that protein production is active in all threestrains. FIG. 1D shows selective detection in the diploid strain ofcorrectly assembled full antibody, confirming that only the diploidsystem is capable of generating fully assembled antibody.

FIG. 2. Full length antibody production in P. pastoris. Heterologousexpression of full-length antibody was conducted using a diploid P.pastoris strain. Exported antibody protein was isolated from conditionedmedium using Protein A affinity chromatography. An aliquot of the peakfraction is shown. The human IgG standard was derived from purifiedpooled human IgG.

FIG. 3. Assembled antibody was detected and characterized from mediasupernatants from subclones of diploid P. pastoris strains, which wereengineered to produce full-length mouse/human chimeric antibody.Microtiter plates were coated with Anti-human Fc selective antibodies tocapture the antibody from the culture media. Correctly assembledantibody was detected through the use of a human selective (Fab′)2,which recognized the paired heavy CH1 and κ light chain constantregions. Serial dilutions of clarified medium were applied to the plate.Development was through standard ELISA visualization methods. Thedetection is selective as shown by the lack of any detectable signal inthe mIgG standard.

FIG. 4. P. pastoris generated recombinant antibody stains CD3 containingJurkat T-cells as well as traditional mammalian-derived antibody. JurkatT-cells were immobilized on glass slides and staining was conductedusing the anti-CD3 antibody generated in yeast and mammalian cells.Detection was performed using a biotinylated-conjugated anti-rodentsecondary antibody, and developed with an HRP-streptavidin derivative.The images are representative field of a slide treated with eachrecombinant antibody. Background is control for development andconducted in the absence of the primary anti-CD3 antibody.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present Assignee, Alder Biopharmaceuticals Inc., has developedmethods for using polyploidal P. pastoris and other yeast species into arobust scalable platform for industrial scale production of heterologousproteins for commercial applications. These methods are genericallyapplicable for expressing any desired protein. However, in preferredembodiments this expression system is utilized to produce mammalianproteins, especially mammalian polypeptides such as multichain proteinshaving therapeutic or diagnostic applications, such as antibodies,enzymes, hormones, growth factors, cytokines, and the like. It has beensurprisingly discovered that polyploidal yeast express heterologousproteins, including multichain proteins such as immunoglobulins forprolonged time periods and at very high yields.

While these methods are described and exemplified in the Assignee'searlier patent applications, it is desired to further enhance thesemethods. Along these lines it is a continuing objective of the Assigneeto identify regulatory sequences and specific mutants or culture methodsthat provide for even higher protein yields or which are desirable fromother perspectives such as enhanced stability. There is a specific needto expand the repertoire of promoters available for use in P. pastoristhat provide for inducible gene expression. Current inducible promotersystems employ materials that pose significant hazards at the scale-upstage. The present invention as described in detail herein provides aseries of novel inducible promoters that employ inexpensive,non-hazardous induction alternatives to the currently available P.pastoris promoter induction systems. Particularly, the present inventionidentifies three novel regulatory sequences derived from P. pastorisgenes, i.e., ADH1 or alcohol dehydrogenase 1, glycerol kinase (GUT1) andenolase (ENO1). The sequences for these three novel promoters areprovided infra.

These novel promoters are desirably used to effect the expression ofrecombinant hetero-multimeric proteins, which are preferably secretedfrom diploid strains of mating competent yeast. In this preferredembodiment of the invention a pair of genetically marked yeast haploidcells are transformed with expression vectors comprising subunits of theheteromultimeric protein operably linked to a novel promoter accordingto the invention. One haploid cell comprises a first expression vector,and a second haploid cell comprises a second expression vector whereineither or both may contain a novel promoter according to the invention.Optionally, additional expression vectors may be introduced into thehaploid or diploid cells; or the first or second expression vectors maycomprise additional coding sequences; for the synthesis ofheterotrimers; heterotetramers; etc. The expression levels of thenon-identical polypeptides may be individually calibrated, and adjustedthrough appropriate selection, vector copy number, promoter strengthand/or induction and the like. The transformed haploid cells aregenetically crossed or fused. The resulting diploid or tetraploidstrains are utilized to produce and secrete fully assembled andbiologically functional hetero-multimeric protein under the regulatorycontrol of at least one promoter according to the invention.

As disclosed in the Assignee's earlier patent applications, the use ofdiploid or tetraploid cells for protein production provides forunexpected benefits. The cells can be grown for production purposes,i.e. scaled up, and for extended periods of time, in conditions that canbe deleterious to the growth of haploid cells, which conditions mayinclude high cell density; growth in minimal medium; growth at lowtemperatures; stable growth in the absence of selective pressure; andwhich may provide for maintenance of heterologous gene sequenceintegrity and maintenance of high level expression over time. Thesebenefits may arise, at least in part, from the creation of diploidstrains from two distinct parental haploid strains. Such haploid strainscan comprise numerous minor autotrophic mutations, which mutations arecomplemented in the diploid or tetraploid, enabling growth under highlyselective conditions.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention which will be limited only by the appended claims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisinvention belongs unless clearly indicated otherwise.

Mating competent yeast species. Such species of yeast exist in a haploidand a diploid form. The diploid cells may, under appropriate conditions,proliferate for indefinite number of generations in the diploid form.Diploid cells can also sporulate to form haploid cells. In addition,sequential mating can result in tetraploid strains through furthermating of the auxotrophic diploids.

In one embodiment of the invention, the mating competent yeast is amember of the Saccharomycetaceae family, which includes the generaArxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera;Eremothecium; lssatchenkia; Kazachstania; Kluyveromyces; Kodamaea;Lodderomyces; Pachysolen; P. pastoris; Saccharomyces; Saturnispora;Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces.

The genus P. pastoris is of particular interest. P. pastoris comprises anumber of species, including the species P. pastoris pastoris, P.pastoris methanolica, and Hansenula polymorpha (P. pastoris angusta).Most preferred is the species P. pastoris.

Haploid Yeast Cell: A cell having a single copy of each gene of itsnormal genomic (chromosomal) complement.

Diploid Yeast Cell: A cell having two copies (alleles) of every gene ofits normal genomic complement, typically formed by the process of fusion(mating) of two haploid cells.

Tetraploid Yeast Cell. A cell having four copies (alleles) of every geneof its normal genomic complement, typically formed by the process offusion (mating) of two haploid cells. Tetraploids may carry two, three,or four different cassettes. Such tetraploids might be obtained in S.cerevisiae by selective mating of homozygotic heterothallic a/a andalpha/alpha diploids and in P. pastoris by sequential mating of haploidsto obtain auxotrophic diploids. For example, a [met his] haploid can bemated with [ade his] haploid to obtain diploid [his]; and a [met arg]haploid can be mated with [ade arg] haploid to obtain diploid [arg];then the diploid [his]×diploid [arg] to obtain a tetraploid prototroph.It will be understood by those of skill in the art that reference to thebenefits and uses of diploid cells may also apply to tetraploid cells.

Yeast Mating: The process by which two haploid yeast cells naturallyfuse to form one diploid yeast cell.

Meiosis: The process by which a diploid yeast cell undergoes reductivedivision to form four haploid spore products. Each spore may thengerminate and form a haploid vegetatively growing cell line.

Selectable Marker: A selectable marker is a gene or gene fragment thatconfers a growth phenotype (physical growth characteristic) on a cellreceiving that gene as, for example through a transformation event. Theselectable marker allows that cell to survive and grow in a selectivegrowth medium under conditions in which cells that do not receive thatselectable marker gene cannot grow. Selectable marker genes generallyfall into several types, including positive selectable marker genes suchas a gene that confers on a cell resistance to an antibiotic or otherdrug, temperature-sensitive (ts) mutants, including both cold-sensitiveand heat-sensitive mutants, e.g., when two ts mutants are crossed or ats mutant is transformed; negative selectable marker genes such as abiosynthetic gene that confers on a cell the ability to grow in a mediumwithout a specific nutrient needed by all cells that do not have thatbiosynthetic gene, or a mutagenized biosynthetic gene that confers on acell inability to grow by cells that do not have the wild type gene; andthe like. Suitable markers and ts mutants are well known and readilyavailable and include but are not limited to: ZEO; G418; HIS 5; LYS3;MET1; MET3a; ADE1; ADE3; URA3; and the like.

Expression Vector: These DNA species contain elements that facilitatemanipulation for the expression of a foreign protein within the targethost cell. Conveniently, manipulation of sequences and production of DNAfor transformation is first performed in a bacterial host, e.g. E. coli,and usually vectors will include sequences to facilitate suchmanipulations, including a bacterial origin of replication andappropriate bacterial selection marker. Selectable markers encodeproteins necessary for the survival or growth of transformed host cellsgrown in a selective culture medium. Host cells not transformed with thevector containing the selection gene will not survive in the culturemedium. Typical selection genes encode proteins that (a) conferresistance to antibiotics or other toxins, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media.

Expression vectors for use in the methods of the invention will furtherinclude yeast specific sequences, including a selectable auxotrophic ordrug marker for identifying transformed yeast strains. A drug marker mayfurther be used to amplify copy number of the vector in a yeast hostcell.

The polypeptide coding sequence of interest is operably linked totranscriptional and translational regulatory sequences that provide forexpression of the polypeptide in yeast cells. These vector componentsmay include, but are not limited to, one or more of the following: anenhancer element, a promoter, and a transcription termination sequence.Sequences for the secretion of the polypeptide may also be included,e.g. a signal sequence, and the like. A yeast origin of replication isoptional, as expression vectors are often integrated into the yeastgenome.

In one embodiment of the invention, the polypeptide of interest isoperably linked, or fused, to sequences providing for optimizedsecretion of the polypeptide from yeast diploid cells.

Nucleic acids are “operably linked” when placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for asignal sequence is operably linked to DNA for a polypeptide if it isexpressed as a preprotein that participates in the secretion of thepolypeptide; a promoter or enhancer is operably linked to a codingsequence if it affects the transcription of the sequence. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading frame. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites oralternatively via a PCR/recombination method familiar to those skilledin the art (Gateway® Technology; Invitrogen, Carlsbad Calif.). If suchsites do not exist, the synthetic oligonucleotide adapters or linkersare used in accordance with conventional practice.

Promoters are untranslated sequences located upstream (5′) to the startcodon of a structural gene (generally within about 100 to 1000 bp) thatcontrol the transcription and translation of particular nucleic acidsequence to which they are operably linked. Such promoters fall intoseveral classes: inducible, constitutive, and repressible promoters thatincrease levels of transcription in response to absence of a repressor.Inducible promoters may initiate increased levels of transcription fromDNA under their control in response to some change in cultureconditions, e.g., the presence or absence of a nutrient or a change intemperature.

The yeast promoter fragment may also serve as the site for homologousrecombination and integration of the expression vector into the samesite in the yeast genome; alternatively a selectable marker is used asthe site for homologous recombination. P. pastoris transformation isdescribed in Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385.

Examples of previously known promoters from P. pastoris useful forheterologous gene expression include the AOX1 promoter (Cregg et al.(1989) Mol. Cell. Biol. 9:1316-1323); ICL1 promoter (Menendez et al.(2003) Yeast 20(13):1097-108); glyceraldehyde-3-phosphate dehydrogenasepromoter (GAP) (Waterham et al. (1997) Gene 186(1):37-44); and FLD1promoter (Shen et al. (1998) Gene 216(1):93-102). The GAP promoter is astrong constitutive promoter and the AOX and FLD1 promoters areinducible.

As previously noted this invention provides three novel promotersequences derived from the ADH1, ENO1 and GUT1 genes of P. pastoris. Itshould be understood that these promoters may comprise the specificnucleic acid sequences provided herein, as well as fragments, mutantsand chimeric prompters derived using these novel promoter sequences. Forexample, the subject promoter sequences may be truncated and thefragments screened to assess those truncated sequences still provide fortranscription of a gene operably linked thereto. Alternatively different5′ portions of the promoter regions described herein may be directly orindirectly linked to other promoters, especially strong constitutivepromoters in order to obtain a chimeric or hybrid promoter that isrendered inducible under the conditions that the ADH1, ENO1 or GUT1promoter is inducible. Still alternatively the subject promoters may bemutagenized by site specific mutagenesis at one or more sites, e.g.,from 1-50 residues, 1-25 residues, 1-10 residues or 1-5 residues and themutant promoter sequences screened to determine the effect of suchmutations on the levels of expression or induction on structural genesoperably linked thereto For example, these methods may be used toidentify mutant promoters which are stronger relative to the wild-typeADH1, ENO1 or GUT1 promoters, or which are “tighter or less leaky”, i.e.do not turn on unless the induction conditions are present. This may beadvantageous in the situation wherein the expressed heterologous proteinis toxic to the yeast cell it is being expressed in. Also the inventionprovides promoters that hybridize to the subject novel promoters understringent hybridization conditions.

The polypeptides of interest which are operably linked to a promoteraccording to the invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, e.g. a signal sequence or other polypeptide having aspecific cleavage site at the N-terminus of the mature protein orpolypeptide. In general, the signal sequence may be a component of thevector, or it may be a part of the polypeptide coding sequence that isinserted into the vector. The heterologous signal sequence selectedpreferably is one that is recognized and processed through one of thestandard pathways available within the host cell. The S. cerevisiaealpha factor pre-pro signal has proven effective in the secretion of avariety of recombinant proteins from P. pastoris. Secretion signals ofinterest also include mammalian signal sequences, which may beheterologous to the protein being secreted, or may be a native sequencefor the protein being secreted. Signal sequences include pre-peptidesequences, and in some instances may include propeptide sequences. Manysuch signal sequences are known in the art, including the signalsequences found on immunoglobulin chains, e.g. kappa 28 preprotoxinsequence, PHA-E, FACE, human MCP-1, human serum albumin signalsequences, human Ig heavy chain, human Ig κ light chain, and the like.For example, see Hashimoto et. al. Protein Eng 11(2) 75 (1998); andKobayashi et. al. Therapeutic Apheresis 2(4) 257 (1998).

Transcription may be further increased by inserting a transcriptionalactivator sequence into the promoter containing vector. These activatorsare cis-acting elements of DNA, usually about from 10 to 300 bp, whichact on a promoter to increase its transcription. Transcriptionalenhancers are relatively orientation and position independent, havingbeen found 5′ and 3′ to the transcription unit, within an intron, aswell as within the coding sequence itself. The enhancer may be splicedinto the expression vector at a position 5′ or 3′ to the codingsequence, but is preferably located at a site 5′ from the promoter.

Expression vectors used in eukaryotic host cells may also containsequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from 3′ tothe translation termination codon, in untranslated regions of eukaryoticor viral DNAs or cDNAs. These regions contain nucleotide segmentstranscribed as polyadenylated fragments in the untranslated portion ofthe mRNA.

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques orPCR/recombination methods. Isolated plasmids or DNA fragments arecleaved, tailored, and re-ligated in the form desired to generate theplasmids required via recombination methods. For analysis to confirmcorrect sequences in plasmids constructed, the ligation mixtures areused to transform host cells, and successful transformants selected byantibiotic resistance (e.g. ampicillin or Zeocin) where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionendonuclease digestion and/or sequenced.

As an alternative to restriction and ligation of fragments, sitespecific recombination methods based on, for example att sites andrecombination enzymes may be used to insert DNA sequences into a vector.Such methods are described, for example, by Landy (1989) Ann. Rev.Biochem. 58:913-949; and are known to those of skill in the art. Suchmethods utilize intermolecular DNA recombination that is mediated by alambda phage and E. coli-encoded recombination proteins. Recombinationoccurs between specific attachment (att) sites on the interacting DNAmolecules. For a description of att sites see Weisberg and Landy (1983)Site-Specific Recombination in Phage Lambda, in Lambda H, Weisberg, ed.(Cold Spring Harbor, N.Y.: Cold Spring Harbor Press), pp. 211-250. TheDNA segments flanking the recombination sites are switched, such thatafter recombination, the att sites are hybrid sequences comprised ofsequences donated by each parental vector. The recombination can occurbetween DNAs of any topology.

att sites may be introduced into a sequence of interest by ligating thesequence of interest into an appropriate vector; generating a PCRproduct containing att B sites through the use of specific primers;generating a cDNA library cloned into an appropriate vector containingatt sites; and the like.

Folding, as used herein, refers to the three-dimensional structure ofpolypeptides and proteins, where interactions between amino acidresidues act to stabilize the structure. While non-covalent interactionsare important in determining structure, usually the proteins of interestwill have intra- and/or intermolecular covalent disulfide bonds formedby two cysteine residues. For naturally occurring proteins andpolypeptides or derivatives and variants thereof, the proper folding istypically the arrangement that results in optimal biological activity,and can conveniently be monitored by assays for activity, e.g. ligandbinding, enzymatic activity, etc.

In some instances, for example where the desired product is of syntheticorigin, assays based on biological activity will be less meaningful. Theproper folding of such molecules may be determined on the basis ofphysical properties, energetic considerations, modeling studies, and thelike.

The expression host may be further modified by the introduction ofsequences encoding one or more enzymes that enhance folding anddisulfide bond formation, i.e. foldases, chaperonins, etc. Suchsequences may be constitutively or inducibly expressed in the yeast hostcell, using vectors, markers, etc. as known in the art. Preferably thesequences, including transcriptional regulatory elements sufficient forthe desired pattern of expression, are stably integrated in the yeastgenome through a targeted methodology.

For example, the eukaryotic PDI is not only an efficient catalyst ofprotein cysteine oxidation and disulfide bond isomerization, but alsoexhibits chaperone activity. Co-expression of PDI can facilitate theproduction of active proteins having multiple disulfide bonds. Also ofinterest is the expression of BIP (immunoglobulin heavy chain bindingprotein); cyclophilin; and the like. In one embodiment of the invention,each of the haploid parental strains expresses a distinct foldingenzyme, e.g. one strain may express BIP, and the other strain mayexpress PDI.

The terms “desired protein” or “target protein” are used interchangeablyand refer generally to any secreted protein having 2 or morenon-identical polypeptide chains, where such chains are independentlysynthesized, i.e. not resulting from post-translational cleavage of asingle polypeptide chain. The polypeptides are heterologous, i.e.,foreign, to the yeast. Preferably, mammalian polypeptides, i.e.polypeptides encoded in a mammalian genome are used.

In a preferred embodiment, the protein is an antibody. The term“antibody” is intended to include any polypeptide chain-containingmolecular structure with a specific shape that fits to and recognizes anepitope, where one or more non-covalent binding interactions stabilizethe complex between the molecular structure and the epitope. Thearchetypal antibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken,other avians, etc., are considered to be “antibodies.” Numerous antibodycoding sequences have been described; and others may be raised bymethods well-known in the art.

For example, antibodies or antigen binding fragments may be produced bygenetic engineering. In this technique, as with other methods,antibody-producing cells are sensitized to the desired antigen orimmunogen. The messenger RNA isolated from antibody producing cells isused as a template to make cDNA using RT-PCR amplification. A library ofvectors, each containing one heavy chain gene and one light chain generetaining the initial antigen specificity, is produced by insertion ofappropriate sections of the amplified immunoglobulin cDNA into theexpression vectors. A combinatorial library is constructed by combiningthe heavy chain gene library with the light chain gene library. Thisresults in a library of clones which co-express a heavy and light chain(resembling the Fab fragment or antigen binding fragment of an antibodymolecule). The vectors that carry these genes are co-transfected into ahost cell. When antibody gene synthesis is induced in the transfectedhost, the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, or to minimize misfolding by substitution or deletion of one ormore cysteine residues that are not necessary for function. Variants canbe designed so as to retain or have enhanced biological activity of aparticular region of the protein (e.g., a functional domain, catalyticamino acid residues, etc). Variants also include fragments of thepolypeptides disclosed herein, particularly biologically activefragments and/or fragments corresponding to functional domains.Techniques for in vitro mutagenesis of cloned genes are known. Alsoincluded in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.

Chimeric antibodies may be made by recombinant means by combining thevariable light and heavy chain regions (VK and VH), obtained fromantibody producing cells of one species with the constant light andheavy chain regions from another. Typically chimeric antibodies utilizerodent or rabbit variable regions and human constant regions, in orderto produce an antibody with predominantly human domains. The productionof such chimeric antibodies is well known in the art, and may beachieved by standard means (as described, e.g., in U.S. Pat. No.5,624,659, incorporated fully herein by reference).

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody andin certain instances a subset of this foreign CDR sequences. This isaccomplished by carefully examining the sequence of the hyper-variableloops of the variable regions of the monoclonal antibody, and fittingthem to the structure of the human antibody chains. Although faciallycomplex, the process is straightforward in practice. See, e.g., U.S.Pat. No. 6,187,287, incorporated fully herein by reference.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) may be synthesized.“Fragment,” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing avariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities.

Immunoglobulins may be modified post-translationally, e.g. to addchemical linkers, detectable moieties, such as fluorescent dyes,enzymes, substrates, chemiluminescent moieties and the like, or specificbinding moieties, such as streptavidin, avidin, or biotin, and the likemay be utilized in the methods and compositions of the presentinvention.

Preferred Methods of Polypeptide Synthesis According to the Invention

As noted above, the subject novel P. pastoris promoters may be used inany compatible eukaryotic expression system but preferably are used inthe Assignee's proprietary polyploid expression system This system canbe used to produce single or multiple subunit polypeptides. If used toproduce a multiple subunit polypeptide such as an immunoglobulin theexpression system may be carried out as follows:

Transformed mating competent haploid yeast cells provide a geneticmethod that enables subunit pairing of a desired protein. Haploid yeaststrains, preferably two differently marked haploids strains, aretransformed with each of two expression vectors, a first vector todirect the synthesis of one polypeptide chain and a second vector todirect the synthesis of a second, non-identical polypeptide chain. Thetwo haploid strains are mated to provide a diploid host where optimizedtarget protein production can be obtained. However, alternatively andless preferably a single haploid strain may be transformed with twovectors or a single vector that provides for the expression of bothpolypeptide subunits and the resultant transformed haploid mated orfused with another haploid to produce a diploid that expresses themultiple subunit polypeptide.

Optionally, additional non-identical coding sequence(s) are provided.Such sequences may be present on additional expression vectors or in thefirst or the second expression vectors. As is known in the art, multiplecoding sequences may be independently expressed from individualpromoters; or may be coordinately expressed through the inclusion of an“internal ribosome entry site” or “IRES”, which is an element thatpromotes direct internal ribosome entry to the initiation codon, such asATG, of a cistron (a protein encoding region), thereby leading to thecap-independent translation of the gene. IRES elements functional inyeast are described by Thompson et al. (2001) P.N.A.S. 98:12866-12868.

In one embodiment of the invention, antibody sequences are produced incombination with a secretory J chain, which provides for enhancedstability of IgA (see U.S. Pat. Nos. 5,959,177; and 5,202,422).

The two haploid yeast strains are each auxotrophic, and requiresupplementation of media for growth of the haploid cells. The pair ofauxotrophs are complementary, such that the diploid product will grow inthe absence of the supplements required for the haploid cells. Many suchgenetic markers are known in yeast, including requirements for aminoacids (e.g. methionine, lysine, histidine, arginine, etc.), nucleosides(e.g. uracil, adenine, etc.); and the like. Amino acid markers may bepreferred for the methods of the invention.

The two transformed haploid cells may be genetically crossed and diploidstrains arising from this mating event selected by their hybridnutritional requirements. Alternatively, populations of the twotransformed haploid strains are spheroplasted and fused, and diploidprogeny regenerated and selected. By either method, diploid strains canbe identified and selectively grown because, unlike their haploidparents, they do not have the same nutritional requirements. Forexample, the diploid cells may be grown in minimal medium. The diploidsynthesis strategy has certain advantages. Diploid strains have thepotential to produce enhanced levels of heterologous protein throughbroader complementation to underlying mutations, which may impact theproduction and/or secretion of recombinant protein.

In one embodiment of the invention, each of the haploid strains istransformed with a library of polypeptides, e.g. a library of antibodyheavy or light chains. Transformed haploid cells that synthesize thepolypeptides are mated with the complementary haploid cells and maydisplay the protein on their cell surface. This methodology whichessentially comprises the construction and diversification of yeast cellsurface displayed libraries produced by mating has been extensively usedin particular for affinity maturation of Fab antibody fragments. (Seee.g., Blaise L et. al. Gene (2004) 342:211-8; and patents assigned toCambridge Antibody Technology such as U.S. Pat. Nos. 7,063,943;6,916,605; 6,806,079; 6,172,197; 5,969,108; and 5,565,332, all of whichare incorporated by reference in their entireties herein). The resultingdiploid cells are screened for functional protein. The diploid cellsprovide a means of rapidly, conveniently and inexpensively bringingtogether a large number of combinations of polypeptides for functionaltesting. This technology is especially applicable for the generation ofheterodimeric protein products, where optimized subunit synthesis levelsare critical for functional protein expression and secretion. As notedyeast display is well used for affinity maturation of Fab fragments. Inaddition it has recently been shown that Fab antibodies can be displayedon the cell surface of Saccharomyces cerevisiae and that affinitymatured Fab antibody fragments sorted by fluorescent-activated cellsorting of yeast-displayed libraries have been identified. (See, Van denBuecken et al., FEBS Letters 546:288-294 (2003)) This discovery and theknowledge that Fab antibodies are heterodimeric suggests thatindependent repertoires of heavy chain (HC) and light chain (LC) can beconstructed in haploid yeast strains of opposite mating type. Theseseparate repertoires can then be combined by highly efficient yeastmating. Using this approach, the present inventors have rapidlygenerated a naïve human Fab yeast display library of over a billionclones. In addition, utilizing error-prone polymerase chain reaction,the inventors have diversified Fab sequences and generated combinatorialand hierarchal chain shuffled libraries with complexities of up to 5billion clones. These libraries can be selected for higher affinityusing a repetitive process of mating-driven chain shuffling and flowcytometric sorting.

In another embodiment of the invention, the expression level ratio ofthe two subunits is regulated in order to maximize product generation.Heterodimer subunit protein levels have been shown previously to impactthe final product generation (Simmons L C, J Immunol Methods. 2002 May1; 263(1-2):133-47). Regulation can be achieved prior to the mating stepby selection for a marker present on the expression vector. By stablyincreasing the copy number of the vector, the expression level can beincreased. In some cases, it may be desirable to increase the level ofone chain relative to the other, so as to reach a balanced proportionbetween the subunits of the polypeptide. Antibiotic resistance markersare useful for this purpose, e.g. Zeocin resistance marker, G418resistance, etc. and provide a means of enrichment for strains thatcontain multiple integrated copies of an expression vector in a strainby selecting for transformants that are resistant to higher levels ofZeocin or G418. The proper ratio, e.g. 1:1; 1:2; etc. of the subunitgenes may be important for efficient protein production. Even when thesame promoter is used to transcribe both subunits, many other factorscontribute to the final level of protein expressed and therefore, it canbe useful to increase the number of copies of one encoded gene relativeto the other. Alternatively, diploid strains that produce higher levelsof a polypeptide, relative to single copy vector strains, are created bymating two haploid strains, both of which have multiple copies of theexpression vectors.

Host cells are transformed with the above-described expression vectors,mated to form diploid strains, and cultured in conventional nutrientmedia modified as appropriate for the subject novel inducible promoters,selecting transformants or amplifying the genes encoding the desiredsequences. As described infra in the examples the subject novelinducible promoters are strongly inducible in the presence of at leastone of glycerol and ethanol and/or repressed by methanol. The inductionmedium will therefore contain these carbon sources or another carbonsource that similarly induces the promoter. With respect thereto anumber of minimal media suitable for the growth of yeast are known inthe art. Any of these media may be supplemented as necessary with salts(such as sodium chloride, calcium, magnesium, and phosphate), buffers(such as HEPES), nucleosides (such as adenosine and thymidine),antibiotics, trace elements, and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Secreted proteins are recovered from the culture medium. A proteaseinhibitor, such as phenyl methyl sulfonyl fluoride (PMSF) may be usefulto inhibit proteolytic degradation during purification, and antibioticsmay be included to prevent the growth of adventitious contaminants. Thecomposition may be concentrated, filtered, dialyzed, etc., using methodsknown in the art.

The diploid cells of the invention are grown for production purposes.Such production purposes desirably include growth in minimal media,which media lacks pre-formed amino acids and other complex biomolecules,e.g. media comprising ammonia as a nitrogen source, and glucose as anenergy and carbon source, and salts as a source of phosphate, calciumand the like. Preferably such production media lacks selective agentssuch as antibiotics, purines, pyrimidines, etc. The diploid cells can begrown to high cell density, for example at least about 50 g/L; moreusually at least about 100 g/L; and may be at least about 300, about400, about 500 g/L or more. If the heterologous protein is potentiallyadverse to yeast growth it may be desirable to initially grow the cellsto high density under non-induction conditions, i.e., the absence ofglycerol or ethanol and thereafter culture the expanded yeast cellsunder induction growth conditions.

In one embodiment of the invention, the growth of the subject cells forproduction purposes is performed at low temperatures, which temperaturesmay be lowered during log phase, during stationary phase, or both. Theterm “low temperature” refers to temperatures of at least about 15° C.,more usually at least about 17° C., and may be about 20° C., and isusually not more than about 25-30° C., more usually not more than about26° C. Growth temperature can impact the production of full-lengthsecreted proteins in production cultures, and decreasing the culturegrowth temperature can strongly enhances the intact product yield. Thedecreased temperature appears to assist intracellular traffickingthrough the folding and post-translational processing pathways used bythe host to generate the target product, along with reduction ofcellular protease degradation.

The methods of the invention provide for expression of secreted, activeprotein, particularly secreted, active antibodies, where “activeantibodies”, as used herein, refers to a correctly folded multimer of atleast two properly paired chains, which accurately binds to its cognateantigen. Expression levels of active protein are usually at least about50 mg/liter culture, more usually at least about 100 mg/liter,preferably at least about 500 mg/liter, and may be 1000 mg/liter ormore.

The methods of the invention can provide for increased stability of thehost and heterologous coding sequences during production. The stabilityis evidenced, for example, by maintenance of high levels of expressionover time, where the starting level of expression is decreased by notmore than about 20%, usually not more than 10%, and may be decreased bynot more than about 5% over about 20 doublings, 50 doublings, 100doublings, or more.

The strain stability is believed to also provide for maintenance ofheterologous gene sequence integrity over time, where the sequence ofthe active coding sequence and requisite transcriptional regulatoryelements are maintained in at least about 99% of the diploid cells,usually in at least about 99.9% of the diploid cells, and preferably inat least about 99.99% of the diploid cells over about 20 doublings, 50doublings, 100 doublings, or more. Preferably, substantially all of thediploid cells will maintain the sequence of the active coding sequenceand requisite transcriptional regulatory elements.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention, which will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the celllines, constructs, and methodologies that are described in thepublications, which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

EXPERIMENTAL Example 1

To demonstrate the efficacy of the diploid expression method of thepresent invention for expressing an antibody the following reagents wereprepared. It should be understood that these methods may be varied bythe substitution of the conventional yeast promoters used therein (e.g.from the GAP and AOX1) with at least one of the novel inducible P.pastoris pastoris promoters from the ADH1, ENO1 and GUT1 genes disclosedin the Example which follows. This Example is intended therefore to beillustrative of the preferred diploid expression system and not of theefficacy of these novel promoters.

Antibody genes: Genes were cloned and constructed that directed thesynthesis of three forms of a chimeric humanized mouse monoclonalantibody OKT3. The sources of the variable regions for use in theseconstructs can be found in Genbank. Accession number A22261; mouse OKT3heavy chain (International Patent Application WO 9109967-A 3 11 Jul.1991). Accession number A22259; mouse OKT3 light chain (InternationalPatent Application WO 9109967-A 3 11 Jul. 1991).

All three forms utilized the identical V_(κ)C_(κ) light chain gene (SEQID NO: 10). For the three heavy chain genes, all encoded the identicalmouse variable region (V_(h)) but differed from each other in the aminoacid sequence of the human heavy chain constant regions. The firstconstruct directed the synthesis of a full-length wild-type heavy chain(C_(γ1)) with its single normal N-linked glycosylation site present(full-length glycosylated heavy chain) (SEQ ID NO: 13 and No 14). Thesecond gene directed the synthesis of a non-glycosylated heavy chaincreated by mutating a nucleotide in the sequence so that a threonine atposition 301 was changed to an alanine in the glycosylation siterecognition sequence (Asn-X-Thr/Ser) (full-length non-glycosylated heavychain) (SEQ ID NO: 15). The third gene construct directed the synthesisof a heavy chain in which most of the constant region was deleted afterthe hinge region (Fab heavy chain) (SEQ ID NO: 16).

Expression vector: The vector contains the following functionalcomponents: 1) a mutant ColE1 origin of replication, which facilitatesthe replication of the plasmid vector in cells of the bacteriumEscherichia coli; 2) a bacterial Sh ble gene, which confers resistanceto the antibiotic Zeocin and serves as the selectable marker fortransformations of both E. coli and P. pastoris; 3) an expressioncassette composed of the glyceraldehyde dehydrogenase gene (GAP gene)promoter, fused to sequences encoding the Saccharomyces cerevisiae alphamating factor pre pro secretion leader sequence, followed by sequencesencoding a P. pastoris transcriptional termination signal from the P.pastoris alcohol oxidase I gene (AOX1). The Zeocin resistance markergene provides a means of enrichment for strains that contain multipleintegrated copies of an expression vector in a strain by selecting fortransformants that are resistant to higher levels of Zeocin.

P. pastoris strains: The auxotrophic strains used for this example arethe P. pastoris ade1 and ura3 strains, which require supplementationwith adenine and uracil, respectively, for growth. Strains met1 and lys3have also been used. Although any two complementing sets of auxotrophicstrains could be used for the construction and maintenance of diploidstrains, these two strains are especially suited for this method for tworeasons. First, they grow more slowly than diploid strains that are theresult of their mating or fusion. Thus, if a small number of haploidade1 or ura3 cells remain present in a culture or arise through meiosisor other mechanism, the diploid strain should outgrow them in culture.The preferred strains for use in the subject P. pastoris diploidexpression system are met1 and lys3.

The second is that it is easy to monitor the sexual state of thesestrains since colonies of the diploid product of their mating are anormal white or cream color, whereas cells of any strains that arehaploid ade1 mutants in a culture form a colony with distinct pink incolor. In addition, any strains that are haploid ura3 mutants areresistant to the drug 5-fluoro-orotic acid (FOA) and can be sensitivelyidentified by plating samples of a culture on minimal medium+uracilplates with FOA. On these plates, only uracil-requiring ura3 mutant(presumably haploid) strains can grow and form colonies. Thus, withhaploid parent strains marked with ade1 and ura3, one can readilymonitor the sexual state of the resulting antibody-producing diploidstrains (haploid versus diploid).

Methods

Construction of pGAPZ-alpha expression vectors for transcription oflight and heavy chain antibody genes. For cloning of both the light andheavy chain variable regions, cells of a mouse OKT3 CD3 hybridoma cellline were grown and total RNA extracted. Two RT-PCR reactions were thenperformed, one specific to light and one specific to heavy chainvariable region encoding sequences of the OKT3 antibody genes. Theprimers employed to amplify out the heavy and light chain variableregion were (SEQ ID NO:1)5′-CCGCTCGAGAAAAGAGAGGCTGAAGCTCAGGTCCAGCTGCAGCAGTC-3′ and (SEQ ID NO:3)5′-CCGCTCGAGAAAAGAGAGGCTGAAGCTCAAATTGTTCTCACCCAGTCTCC-3′ along with (SEQID NO:2) 5′-TGGGCCCTTGGTGGAGGCTGAGGAGACTGTGAGAGTGGTGC-3′ and (SEQ IDNO:4) 5′-GACAGATGGTGCAGCCACAGCCCGG TTTATTTCCAACTTTGTCC-3′ for therespective variable regions.

For the human heavy and light chain constant region genes, a humanleukocyte 5′-stretch plus cDNA library was purchased from Clontech (HL5019t). Two PCR reactions were performed on this library using primersspecific for the heavy and light chain constant regions, respectively(Heavy chain: (SEQ ID NO:6)5′-GCACCACTCTCACAGTCTCCTCAGCCTCCACCAAGGGCCCA-3 and (SEQ ID NO:5)5′-ATAAGAATGCGGCCGCTCATTTACCCGGAGACAGGGAG-3′ for full length along with(SEQ ID NO:7) 5′-TGCGGCCGCTCATGGGCACGGTGGGCATGTGT-3′ for FABgeneration′; Light chain: (SEQ ID NO:9)5′-GGACAAAGTTGGAAATAAACCGGGCTGTGGCTGCACCATCTGTC-3′ and (SEQ ID NO:8)5′-ATAAGAATGCGGCCGCTAACACTCTCCCCTGTTGAAGCT-3′.

A DNA sequence encoding the mouse light chain variable region was fusedin frame to a sequence encoding the human light chain constant region(SEQ ID NO: 11 and SEQ ID NO:12). A fragment encoding the final fusionconstruct was inserted into P. pastoris expression vector pGAPZ-alphavia ligation through 5′-Xhol and 3′-Notl sites in pGAPZ-alpha. DNAsequence encoding the mouse heavy variable region was fused in frame tosequences encoding each of the three human heavy chain constant regions.These fusion products were then inserted using a similar 5′-Xhol and3′-Notl strategy into pGAPZ-alpha. (SEQ ID NO:13 and SEQ ID NO: 14 forthe glycosylated version; SEQ ID NO: 15 for the aglycosylated version;SEQ ID NO: 16 for the Fab fragment). The proper antibody gene DNAsequences in all vectors were confirmed by direct DNA sequencing priorto further work.

Transformation of Expression Vectors into Haploid ade1 ura3, met1 andlys3 Host Strains of P. pastoris.

All methods used for transformation of haploid P. pastoris strains andgenetic manipulation of the P. pastoris sexual cycle were as describedin Higgins, D. R., and Cregg, J. M., Eds. 1998. P. pastoris Protocols.Methods in Molecular Biology. Humana Press, Totowa, N.J.

Prior to transformation, each expression vector was linearized withinthe GAP promoter sequences with AvrII to direct the integration of thevectors into the GAP promoter locus of the P. pastoris genome. Samplesof each vector were then individually transformed into electrocompetentcultures of the ade1, ura3, met1 and lys3 strains by electroporation andsuccessful transformants were selected on YPD Zeocin plates by theirresistance to this antibiotic. Resulting colonies were selected,streaked for single colonies on YPD Zeocin plates and then examined forthe presence of the antibody gene insert by a PCR assay on genomic DNAextracted from each strain for the proper antibody gene insert and/or bythe ability of each strain to synthesize an antibody chain by a colonylift/immunoblot method (Wung et. al. Biotechniques 21 808-812 (1996).Haploid ade1, met1 and lys3 strains expressing one of the three heavychain constructs were collected for diploid constructions along withhaploid ura3 strain expressing light chain gene. The haploid strainsexpressing each of the heavy chain genes were mated with the appropriatelight chain haploid ura3 strain to generate diploid secreting protein.

Mating of Haploid Strains Synthesizing a Single Antibody Chain andSelection of Diploid Derivatives Synthesizing Tetrameric FunctionalAntibodies.

To mate P. pastoris haploid strains, each ade1 (or meta or lys3) heavychain producing strain to be crossed was streaked across a rich YPDplate and the ura3 light chain producing strain was streaked across asecond YPD plate (˜10 streaks per plate). After one or two daysincubation at 30° C., cells from one plate containing heavy chainstrains and one plate containing ura3 light chain strains weretransferred to a sterile velvet cloth on a replica-plating block in across hatched pattern so that each heavy chain strain contained a patchof cells mixed with each light chain strain. The cross-streaked replicaplated cells were then transferred to a mating plate and incubated at25° C. to stimulate the initiation of mating between strains. After twodays, the cells on the mating plates were transferred again to a sterilevelvet on a replica-plating block and then transferred to minimal mediumplates. These plates were incubated at 30° C. for three days to allowfor the selective growth of colonies of prototrophic diploid strains.Colonies that arose were picked and streaked onto a second minimalmedium plate to single colony isolate and purify each diploid strain.The resulting diploid cell lines were then examined for antibodyproduction.

Putative diploid strains were tested to demonstrate that they werediploid and contained both expression vectors for antibody production.For diploidy, samples of a strain were spread on mating plates tostimulate them to go through meiosis and form spores. Haploid sporeproducts were collected and tested for phenotype. If a significantpercentage of the resulting spore products were single or doubleauxotrophs we concluded that the original strain must have been diploid.Diploid strains were examined for the presence of both antibody genes byextracting genomic DNA from each and utilizing this DNA in PCR reactionsspecific for each gene.

Fusion of Haploid Strains Synthesizing a Single Antibody Chain andSelection of Diploid Derivatives Synthesizing Tetrameric FunctionalAntibodies.

As an alternative to the mating procedure described above, individualcultures of single-chain antibody producing haploid ade1 and ura3strains were spheroplasted and their resulting spheroplasts fused usingpolyethylene glycol/CaCl₂. The fused haploid strains were then embeddedin agar containing 1 M sorbitol and minimal medium to allow diploidstrains to regenerate their cell wall and grow into visible colonies.Resulting colonies were picked from the agar, streaked onto a minimalmedium plate, and the plates incubated for two days at 30° C. togenerate colonies from single cells of diploid cell lines. The resultingputative diploid cell lines were then examined for diploidy and antibodyproduction as described above.

Purification and Analysis of Antibodies.

A diploid strain for the production of full length antibody was derivedthrough the mating of ura3 light chain strain 2252 and lys3 heavy chainstrain 2254 using the methods described above. Culture media fromshake-flask or fermenter cultures of diploid P. pastoris expressionstrains were collected and examined for the presence of antibody proteinvia SDS-PAGE and immunoblotting using antibodies directed against heavyand light chains of human IgG, or specifically against the heavy chainof IgG. The data is shown in FIG. 2.

To purify the yeast secreted antibodies, clarified media from antibodyproducing cultures were passed through a protein A column and afterwashing with 20 mM sodium phosphate, pH 7.0, binding buffer, protein Abound protein was eluted using 0.1 M glycine HCl buffer, pH 3.0.Fractions containing the most total protein were examined by Coomasieblue strained SDS-PAGE and immunoblotting for antibody protein.Fractions were also examined via an ELISA assay in which microtiterplates were first coated with F(ab′)2 goat anti-human IgG, Fcγ (JacksonImmuno, Cat No. 109-006-008). Next plates were reacted with selecteddilutions of yeast made antibodies. Finally, plates were reacted withHRP-conjugated goat anti-human F(ab′)2 fragment of IgG F(ab′)2 (JacksonImmuno, Cat No. 109-036-097). Plates were then developed with TMPsubstrate (Sigma Chemical) and reactions were quenched with 0.5 M HCl.Results were quantitated on a BioRad microtiter plate reader at 415 nm.The data are illustrated in FIG. 3.

Assay for Antibody Activity.

The recombinant yeast-derived chimeric antibody was evaluated forfunctional activity through immunohistochemical staining of cellscontaining the target antigen. The chimeric antibody selectivelyrecognizes the CD3 complex found on T cells. Jurkat T cells wereemployed as a source of antigen and cell surface staining was conductedusing procedures described in Andersson and Sander (Immunol Lett. 1989Jan. 31; 20(2):115-20) or Andersson et. al. (Eur J Immunol. 1988December; 18(12):2081-4).

Jurkat T cells were immobilized on glass slides, blocked with theappropriate blocking serum and stained with mammalian and yeastgenerated recombinant primary antibody for 1 hour. The immobilizedsamples were then treated with peroxidase blocking agent followed bystaining with a biotinylated Fc selective secondary antibody that isspecific for each form of the antibody (anti-mouse for the mammalian andanti-human for the yeast). Detection was performed using aHRP-Streptavidin system. Digital imaging was performed to collect thedata for each stained sample. Positive signal is detected in samples viaa dark staining of the cells observed in the panels formammalian-derived and yeast-derived OKT-3. This is data is shown in FIG.4.

Example 2 Identification of Novel P. pastoris Promoters

Objective:

P. pastoris has been developed into a robust scalable platform forindustrial scale production of heterologous proteins for commercialapplications. As noted previously, there is a need to expand the geneexpression technology in this yeast strain for specific expressioninduction control of heterologous protein production in P. pastoris.Current inducible promoter systems employ materials that posesignificant hazards at the scale-up stage. These studies were designedto investigate alternate regulated promoters in P. pastoris employinginexpensive, non-hazardous induction alternatives. Experimental data hasnow identified a series of novel promoters that operate using inductionagents that fit this criterion.

Concept:

It is known from published data that some S. cerevisiae genes whoseproducts are involved in central carbohydrate metabolism exhibit highexpression levels under particular growing conditions and repressedunder others. For example, many enzymes involved in gluconeogenesis arereduced in presence of glucose and dramatically increased when glucoseis replaced by alternative carbon sources. This repression/activation isregulated at the transcriptional level and is controlled by thespecificity of their promoters. Based on this conjecture, we haveselected a number of P. pastoris genes that we expect may exhibit strongregulation (see Table 1).

TABLE 1 Gene Selection Level of induction P. pastoris gene EnzymeEncoded in S. cerevisiae PP-ACS1A Acetyl-CoA synthetase ×13   PP-ACS1BAcetyl-CoA synthetase ×3   PP-FPB1 Fructose-1,6-phosphatase ×14.4PP-ADH1 Alcohol dehydrogenase PP-ADH2 Alcohol dehydrogenase PP-PCK1APhosphoenolpyruvate carboxykinase ×14.7 PP-PCK1B Phosphoenolpyruvatecarboxykinase ×14.7 PP-PYC1A Phosphoenolpyruvate carboxykinase ×14.7PP-HXK1 Hexokinase ×5.8  PP-PGI1 Glucose-6-phosphate isomerase PP-PYC1Pyruvate carboxylase ×3.1  PP-GUT1 Glycerol kinase PP-GUT2Glycerol-3-phosphate dehydrogenase PP-ALD2 Aldehyde dehydrogenase ×12.4PP-ALD6 Aldehyde dehydrogenase ×12.4 (ALD2C) PP-PGM2 Phosphoglucomutase×9.1  PP-GSH2 Glutathione synthetase constitutive PP-ENO1 Enolase ×3  PP-PDC6 Pyruvate decarboxylase ×12  

A second potential approach in identifying a strongly expressing andregulated promoter in P. pastoris is based on comparison of expressionlevels of cellular proteins in the wild type P. pastoris growing onselected carbon sources. Cell free extracts were obtained from themid-logarithmic phase cells grown in liquid media with methanol,ethanol, glycerol or glucose. The proteins from each sample wereseparated by PAGE, transferred to a membrane, visualized by Coommassieblue staining and major protein bands that presumably representedproteins synthesized by higher expressing genes were analyzed byN-terminal sequencing. The resulting sequences were then analyzed. Onenotable result was the presence of a 46.5 kDa protein that is highlyexpressed in all but methanol medium which was identified as Eno1p(Enolase). A second 37 kDa protein representing a major protein inducedby methanol was identified as NAD-dependent Formate Dehydrogenase(Fdh1p), and lastly a 58 kDa protein expressed at high levels in mediumwith ethanol was identified as Myo-Inositol-1-Phosphate Synthase(Ino1p).

These two strategies provided the genes that were used to interrogatethe genomic sequence of P. pastoris to locate the appropriate promoterregions. From this bioinformatics strategy, molecular biology reagentswere generated to recover upstream potential regulatory regions of thesegenes through a PCR-based approach.

Cloning of Promoters and Construction of Test Plasmids with b-LactamaseReporter Gene

Five genes were chosen for further studies based on Table 1: GSH2, ENO1,PDC6, GUT1 and ADH1. As a reporter gene, a modified E. coli®-lactamasegene with Zeo gene as selective marker in the vector backbone waschosen. This vector pPIC-bla can be easily modified through theinsertion of a fragment using the 5′ BglII and 3′BstBI cloning sites todrive β-lactamase expression. To introduce chosen DNA fragments into thevector, 5′ primers containing sequences either for BamHI or BglII and 3′primers with NarI, BstBI or ClaI were designed. Each primer containedabout 20 bp of sequence complementary to sequences 5′ of the ORF of eachgene. Typically constructs contain approximately 1000 bp of sequenceupstream of the ORF. Using this strategy, P. pastoris genomic DNA wasused as template and all five promoters were amplified by PCR followedby cloning into a TOPO vector. After sequencing to confirm that thecorrect sequences of the cloned fragments and presence of the cloningsites, the promoter test fragments were subcloned into BglII andBstBI-digested vector pPIC-bla. The correct structures of plasmidsdesignated as pGSH2-blaZ, pENO1-blaZ, pPDC6-blaZ, pGUT1-blaZ andpADH1-blaZ were confirmed by PCR and DNA sequencing. All plasmids werelinearized by digestion within a unique site located in the clonedfragments (promoters) and introduced by electroporation into wild typeP. pastoris strain Y-11430. Zeocin resistant colonies were selected andanalyzed by PCR for the presence of the hybrid (promoter-reporter gene)DNA. Positive transformants were also analyzed for expression ofβ-lactamase. As positive controls, transformants with pPICZ-bla(=pAOX1-blaZ: high expression of β-lactamase in methanol) and pGAP-blaZ(high constitutive expression of -lactamase) were used. As a negativecontrol, a transformant of pPICZ-HSA expressing another protein undercontrol of the AOX1 promoter was used.

Representative Example of Promoter Cloning

Waterham et al. (Gene 186, 1997) created the plasmid pHWO19A encoding anN-terminally truncated b-lac protein (gene designation: bla). The aminoacid sequence of the modified bla gene has a 23 amino acid deletion andencodes amino acids from His₂₄ through Trp₂₈₆ (Sutcliffe, 1978) precededby Met-Ser-Gly. This modified bla gene was introduced into pPICZ toobtain the bla gene under control of the AOX1 promoter.

The nucleotide sequence of the P. pastoris ADH1 promoter was amplifiedby PCR using P. pastoris genomic DNA as template and two primers 5IT221and 3IT222:

(SEQ ID NO: 16) 5IT221: 5′-GGATCCTTTTTACCACCCAAGTGC-3′ (SEQ ID NO: 17)3IT222: 5-ATCGATAAAAGCTAGTAGCTGATGGAAGAA-3′

The resulting 1033 bp PCR product was digested with BamHI and ClaI andligated into vector pPICZ-bla digested with BglII and BstBI to replacethe AOX1 promoter.

The nucleotide sequence of the P. pastoris ADH1 gene (SEQ ID NO:18)(promoter region intervenes the asterisked regions of the gene sequence,the remaining residues are the putative ORF):

(SEQ ID NO: 18) ***TCCTTTTTACCACCCAAGTGCGAGTGAAACACCCCATGGCTGCTCTCCGATTGCCCCTCTACAGGCATAAGGGTGTGACTTTGTGGGCTTGAATTTTACACCCCCTCCAACTTTTCTCGCATCAATTGATCCTGTTACCAATATTGCATGCCCGGAGGAGACTTGCCCCCTAATTTCGCGGCGTCGTCCCGGATCGCAGGGTGAGACTGTAGAGACCCCACATAGTGACAATGATTATGTAAGAAGAGGGGGGTGATTCGGCCGGCTATCGAACTCTAACAACTAGGGGGGTGAACAATGCCCAGCAGTCCTCCCCACTCTTTGACAAATCAGTATCACCGATTAACACCCCAAATCTTATTCTCAACGGTCCCTCATCCTTGCACCCCTCTTTGGACAAATGGCAGTTAGCATTGGTGCACTGACTGACTGCCCAACCTTAAACCCAAATTTCTTAGAAGGGGCCCATCTAGTTAGCGAGGGGTGAAAAATTCCTCCATCGGAGATGTATTGACCGTAAGTTGCTGCTTAAAAAAAATCAGTTCAGATAGCGAGACTTTTTTGATTTCGCAACGGGAGTGCCTGTTCCATTCGATTGCAATTCTCACCCCTTCTGCCCAGTCCTGCCAATTGCCCATGAATCTGCTAATTTCGTTGATTCCCACCCCCCTTTCCAACTCCACAAATTGTCCAATCTCGTTTTCCATTTGGGAGAATCTGCATGTCGACTACATAAAGCGACCGGTGTCCGAAAAGATCTGTGTAGTTTTCAACATTTTGTGCTCCCCCCGCTGTTTGAAAACGGGGGTGAGCGCTCTCCGGGGTGCGAATTCGTGCCCAATTCCTTTCACCCTGCCTATTGTAGACGTCAACCCGCATCTGGTGCGAATATAGCGCACCCCCAATGATCACACCAACAATTGGTCCACCCCTCCCCAATCTCTAATATTCACAATTCACCTCACTATAAATACCCCTGTCCTGCTCCCAAATTCTTTTTTCCTTCTTCCATCAGCTACTAGCTTTTATCTTATTTACTTTACGAAA***ATGTCTCCAACTATCCCAACTACACAAAAGGCTGTTATCTTCGAGACCAACGGCGGTCCCCTAGAGTACAAGGACATTCCAGTCCCAAAGCCAAAGTCAAACGAACTTTTGATCAACGTTAAGTACTCCGGTGTCTGTCACACTGATTTGCACGCCTGGAAGGGTGACTGGCCATTGGACAACAAGCTTCCTTTGGTTGGTGGTCACGAAGGTGCTGGTGTCGTTGTCGCTTACGGTGAGAACGTCACTGGATGGGAGATCGGTGACTACGCTGGTATCAAATGGTTGAACGGTTCTTGTTTGAACTGTGAGTACTGTATCCAAGGTGCTGAATCCAGTTGTGCCAAGGCTGACCTGTCTGGTTTCACCCACGACGGATCTTTCCAGCAGTATGCTACTGCTGATGCCACCCAAGCCGCCAGAATTCCAAAGGAGGCTGACTTGGCTGAAGTTGCCCCAATTCTGTGTGCTGGTATCACCGTTTACAAGGCTCTTAAGACCGCTGACTTGCGTATTGGCCAATGGGTTGCCATTTCTGGTGCTGGTGGAGGACTGGGTTCTCTTGCCGTTCAATACGCCAAGGCTCTGGGTTTGAGAGTTTTGGGTATTGATGGTGGTGCCGACAAGGGTGAATTTGTCAAGTCCTTGGGTGCTGAGGTCTTCGTCGACTTCACTAAGACTAAGGACGTCGTTGCTGAAGTCCAAAAGCTCACCAACGGTGGTCCACACGGTGTTATTAACGTCTCCGTTTCCCCACATGCTATCAACCAATCTGTCCAATACGTTAGAACTTTGGGTAAGGTTGTTTTGGTTGGTCTGCCATCTGGTGCCGTTGTCAACTCTGACGTTTTCTGGCACGTTCTGAAGTCCATCGAGATCAAGGGATCTTACGTTGGAAACAGAGAGGACAGTGCCGAGGCCATCGACTTGTTCACCAGAGGTTTGGTCAAGGCTCCTATCAAGATTATCGGTCTGTCTGAACTTGCTAAGGTCTACGAACAGATGGAGGCTGGTGCCATCATCGGTAGATACGTTGTGGACACTTCC AAATAA

The putative amino acid sequence of the P. pastoris ADH1 protein is:

(SEQ ID NO: 19) MSPTIPTTQKAVIFETNGGPLEYKDIPVPKPKSNELLINVKYSGVCHTDLHAWKGDWPLDNKLPLVGGHEGAGVVVAYGENVTGWEIGDYAGIKWLNGSCLNCEYCIQGAESSCAKADLSGFTHDGSFQQYATADATQAARIPKEADLAEVAPILCAGITVYKALKTADLRIGQWVAISGAGGGLGSLAVQYAKALGLRVLGIDGGADKGEFVKSLGAEVFVDFTKTKDVVAEVQKLTNGGPHGVINVSVSPHAINQSVQYVRTLGKVVLVGLPSGAVVNSDVFWHVLKSIEIKGSYVGNREDSAEAIDLFTRGLVKAPIKIIGLSELAKVYEQMEAGAIIGRYVVDTSK

The subsequent promoter constructs were generated in a similar fashionusing the sequences demarked below specific to each promoter fragment.Note: Primers used to amplify from the 5′ and 3′ ends of the genomicsequence are found at the start of each section.

ENO1: (SEQ ID NO: 20) 5IT215: 5′-AGATCTGGGCAAAATCACACAATTC-3′(SEQ ID NO: 21) 3IT216: 5′-TTCGAATTGTTATAATTGTGTGTTTCAACCAAG-3′(SEQ ID NO: 22) AGATCTGGGCAAAATCACACAATTCCAAACCATGCTAAATGAGATTTAAAGAACAAACGATGGCAAAAGGCAACCGTTATAAATGTGATCTTTCTTGGCAGTTATCTGTCAATTTTTCTAAGGAACAGTGAATTCATCATAGGAGAGATGTTATACGTTACATAATCATACATACTGCATGTATCTCACCTACTTTACCTCATCAACTCTAAAACAGTTCTAGTCCCAACCCCAGATTCCTAGTCATGACACAAGTCCGCACCGGACAGGACTCACAACCAGCAAGAGAAGCTAACAAATTTACGCCCCGGTAAAACATTCCTTAGGGGCCGTTCAATGGTAATTTTCCTCTCACCCGTTTAAACTTACCTCCGGGCGGTATCTTCAATAACCTCTGTTGTCCCCGGGTATCATTGGAAACAGTGAGGGACGTTGAACAGAAGAGAGGATCACCGTAAATTTGCCTTGCAATTGGCCCTAACCACGGATGGTTAACTTCAAGCCATCACGACAGCAATTGAGTCGGCGCATAGCTACCCTCCTCTTCTTGACCCCATGCATAGGACCAACCTTAACCGATGGAACAGGTTCCTCCGCTCCGTCCCCTGGTAGTGTCTCTGCGCAAGAAATAGTTAAGGTATGAAGACTGATCTCTCGCACCCCCCTCACAGTACTGTTATGGTGAATTGACAAAGCCATTGGCTAGATTGAAACATGTAATTCATATGTAATCTTGTTCAATTAACGAGCTTCGTACAGTCTCAATCTAGACGTCTGATAATGGCGTTTGTGCTCCTAATCGATGAGCCATCTCATGTGACGTCTATACGCTTCGATGGCTTCCGTCGCGAATATAGAACCACTTGAAATATGCTGCAAACCACGATCCACCCTGGTCCTGAAAAGATATAAATACAGCACATCTAGCAGGCTTTTGTCTTCTTGGTTGAAACACACAATTATAACAATTCGAA PDC6: (SEQ ID NO: 23)5IT217: 5′-AGATCTAAAGCATTGCTGAGCAATATTTC-3′ (SEQ ID NO: 24)3IT218: 5′-ATCGATGTACTAGCTAATTGATTGATGATTAACG-3′ (SEQ ID NO: 25)AGATCTAAAGCATTGCTGAGCAATATTTCGGATCAACATCAACAGAATAGATCGTCACCACGAAGTATATCCAACTATTCCCCAAGAATCCAGGCTTATCCCTCAAGAATGGCCTCTCCATCTCCTTCAATGAAGATGGCATTGACAAACTCCGTCAGCTTGCAGGATACAATGGATGCGGAACTAGAAGCGTTGGCAAACGAACAATATTACGTCATGCTAGATATCCTTAAGGGATTTTGCGATCTTTCATTTGACATGGTAAACATATTTTCTATCCAGATCCCTGAGGTATTACATCTCCTCTTCGGCTTTGGTGCAGGGTCACTGGCCTTAACGGGAGTCGTATCCAAAACGAGACAAGAACTAATAAATAAACAGATATAAGGGACAAGCACACGATTACCCAATCACTTGATATGCACCAATTTGTTCCGTTGTTTATGCCATATTTACCGAATTTTCTTCCCAGGTTTTTCCGAATGGACATCTGTAGTCCACTTTTTGGTTATCATAATCGTCCCACAAGTCGTGGATTTAACCAGAACCTAGTAATTTTAAGTTCGCTATTAATCACTCAGAATGGTCTCACCTTGCTATTGGCCAAGTCTGGAGTCGCCAGCTACCACCTCAGAGGCTACATAGACCTCCCAATGTCATCTCCTCAGTGCGCTCTTCAATCTCGTGTCTTTTCCGTTAAAACTCCGTTCGTTTCACCCTATACTGCCCCTGGTTGTGCAGCTCTTACCACTTCGCGCCGCTACTATCCGTAGTGGTCGAGCCGCATCAATATCACGTTGAAATAGAATAACTCCCTACAAAAGCCGCACGCAACCATCAAATCTATATAAGGAACCTCAAATATCTAGCAACATCTTTTCAATTTACTACAACATATTCGTTAATCATCAATCAATTAGCTAGTACATCGAT GUT1: (SEQ ID NO: 26)5IT219: 5′-AGATCTTTCAAGTCGGACCCCAACT-3′ (SEQ ID NO: 27)3IT220: 5′-GGCGCCTGTGGTATAGTGTGAAAAAGTAGAAGAAG-3′ (SEQ ID NO: 28)TTTCAAGTCGGACCCCAACTTTCAAGTGACCCAATTTAGCAGCCTGCATTCTCTTGATTTTATGGGGGAAACTAACAATAGTGTTGCCTTGATTTTAAGTGGCATTGTTCTTTGAAATCGAAATTGGGGATAACGTCATACCGAAAGGTAAACAACTTCGGGGAATTGCCCTGGTTAAACATTTATTAAGCGAGATAAATAGGGGATAGCGAGATAGGGGGCGGAGAAGAAGAAGGGTGTTAAATTGCTGAAATCTCTCAATCTGGAAGAAACGGAATAAATTAACTCCTTCCTGAGATAATAAGATCCGACTCTGCTATGACCCCACACGGTACTGACCTCGGCATACCCCATTGGATCTGGTGCGAAGCAACAGGTCCTGAAACCTTTATCACGTGTAGTAGATTGACCTTCCAGCAAAAAAAGGCATTATATATTTTGTTGTTGAAGGGGTGAGGGGAGGTGCAGGTGGTTCTTTTATTCGTCTTGTAGTTAATTTTCCCGGGGTTGCGGAGCGTCAAAAGTTTGCCCGATCTGATAGCTTGCAAGATGCCACCGCTTATCCAACGCACTTCAGAGAGCTTGCCGTAGAAAGAACGTTTTCCTCGTAGTATTCCAGCACTTCATGGTGAAGTCGCTATTTCACCGAAGGGGGGGTATTAAGGTTGCGCACCCCCTCCCCACACCCCAGAATCGTTTATTGGCTGGGTTCAATGGCGTTTGAGTTAGCACATTTTTTCCTTAAACACCCTCCAAACACGGATAAAAATGCATGTGCATCCTGAAACTGGTAGAGATGCGTACTCCGTGCTCCGATAATAACAGTGGTGTTGGGGTTGCTGTTAGCTCACGCACTCCGTTTTTTTTTCAACCAGCAAAATTCGATGGGGAGAAACTTGGGGTACTTTGCCGACTCCTCCACCATACTGGTATATAAATAATACTCGCCCACTTTTCGTTTGCTGCTTTTATATTTCAAGGACTGAAAAAGACTCTTCTTCTACTTTTTCACACTATACCACAGGCGCCPromoter Induction Studies

The promoter containing strains were evaluated under a series ofconditions to test the selectivity of different glycolytic agents andthe influence these agents have towards the induction of the reportergene. β-lactamase levels are directly proportional to the strength ofinduction by the test agent. A subset of table 1 was explored in thesestudies.

P. pastoris transformants containing the constructions were grown in 50ml buffered (pH6.0) liquid YNB medium with methanol, ethanol, glycerolor glucose for 28 h and samples were collected every 5 h. Cell-freeextracts of each cell sample were prepared and analyzed for proteinconcentration using Pierce BCA reagent and for β-lactamase activityusing PADAC as substrate. The normalization of protein enablesassessment of the efficiency of the induction. The results of thisanalysis are summarized in Table 2.

TABLE 2 Comparison of b-lactamase specific activities in P. pastorisb-lactamase activity compared to activity of pGAP1-bla on glucose at 20h (no—no activity, ww—very weak, w—weak, h—high) Expression Carbonsource Properties of the cassette: Glucose Glycerol Ethanol Methanolpromoter pGSH2-bla w w w w Weak constitutive pADH1-bla w h h ww Strong,induced by glycerol and ethanol pGUT1-bla w h w w Strong, induced byglycerol pENO1-bla h h h w Strong, repressed by methanol pPDC6-bla h h hw Strong, repressed by methanol pGAP1-bla h h h h Strong constitutive,partially repressed by methanol and glycerol pAOX1-bla no ww no hStrong, induced by methanol pAOX1-GSH no no no no

These experimental results highlight the following advantages of theinvention:

-   -   1. P. pastoris ADH1 promoter induction profile is consistent        with the yeast physiology in the two metabolic conditions shown        to have strong enzyme induction and this inductions is        significant with selective carbon sources.    -   2. The GUT1 promoter whose gene encodes glycerol kinase        selectively response to glycerol, which is again consistent with        the metabolic pathway, in which this gene operates.    -   3. The endolase gene (ENO1) promoter responds to broad carbon        sources, which is in line with the central biochemistry this        enzyme plays in carbon metabolism.

CONCLUSIONS AND APPLICATIONS OF THE SUBJECT NOVEL PROMOTERS

To date the dominant regulated promoters employed in the P. pastorisexpression system are from the following genes: AOX1, FLD and GAP. Thethree promoters ADH1, GUT1 and ENO1 are novel and have not previouslybeen characterized from P. pastoris. The AOX1 promoter system is thedominant inducible system in P. pastoris and suffers from the scale-upneed of significant oxygen and methanol feeds in the fermenter. This isan extremely combustible combination and requires extensive control toensure a non-hazardous process. In the case of the FLD promoter system,which has not seen wide implementation, the inducing agent is eithermethanol or a highly volatile toxic alkyl amine. The 3 new promotersoffer pronounced advantages due to the nature of the induction agentsused which are inexpensive as well as non-hazardous in nature. As suchthey possess characteristics important in commercialization processdevelopment. In addition, in the case of the ADH1 promoter there issignificant induction by the promoter for the reporter under conditionswhere elevated ethanol is present either through rich carbon feeding(through the use of glycerol) or direct exposure to alcohol. There areclear feeding strategies during fermentation where this promoter systemcan be kept in a tight off state and then switching to a carbon supportstrategy that initiates bulk product generation under high cell densityconditions. In certain instances depending on the heterologous proteinbeing expressed there can be significant advantages for this level ofcontrol. These range from increased overall protein production, enhancedstability and the ability to successfully generate protein product underconditions in which the expressed protein is toxic.

All references and publications referenced herein are incorporated byreference i their entireties.

In order to better illustrate the invention and its applications thefollowing claims are provided.

What is claimed is:
 1. An isolated nucleic acid sequence containing anenolase (ENOI) promoter derived from the P. pastoris ENOI gene havingthe nucleic acid sequence contained in SEQ ID NO:22, which when operablylinked to a structural gene renders the expression of the gene methanolrepressive, and which is operably linked to a heterologous structuralgene.
 2. The isolated nucleic acid sequence of claim 1 wherein saidstructural gene encodes a non-yeast protein.
 3. The isolated nucleicacid sequence of claim 2 wherein the structural gene is a mammaliangene.
 4. The isolated nucleic acid sequence of claim 2 wherein thestructural gene encodes an antibody polypeptide.
 5. A plasmid containingthe isolated nucleic acid of claim
 1. 6. A plasmid containing theisolated nucleic acid of claim
 2. 7. A plasmid containing the isolatednucleic acid of claim
 3. 8. A plasmid containing the isolated nucleicacid of claim
 4. 9. A yeast cell comprising an isolated nucleic acidaccording to claim 1, wherein the structural gene is other than ENO1.10. A yeast cell comprising an isolated nucleic acid according to claim1, wherein the structural gene is a mammalian structural gene.
 11. Ayeast cell comprising an isolated nucleic acid according to claim 1,wherein the structural gene is a sequence encoding an antibodypolypeptide.
 12. A yeast cell according to claim 9 which is a P.pastoris, Candida, Saccharomyces, Yarrowia, Kluyveromyces, Hansenula, orSchizosaccharomyces yeast cell.
 13. The yeast cell of claim 9 which is aP. pastoris yeast cell.
 14. The yeast cell of claim 12 which ispolyploid.
 15. The yeast cell of claim 13 which is polyploid.
 16. Amethod of using a yeast cell according to claim 9 to produce the proteinencoded by said structural gene under the regulatory control of saidENO1 promoter.
 17. The method of claim 16 wherein the protein is amammalian protein.
 18. The method of claim 17 wherein the mammalianprotein is an antibody, cytokine or growth factor.